Lipogenesis is exquisitely regulated by nutritional/hormonal status. Fatty acid synthase (FAS) is a central enzyme in lipogenesis and is thought to be a rate-limiting step in long-term regulation. FAS transcription, which is low in the fasted state, increases drastically with feeding. Increased insulin is largely responsible for the activation of FAS transcription. During the last grant period, we showed that USF works as a molecular switch by recruiting multiple proteins to the FAS promoter region. In the presence of insulin, USF interacts with DNA-PK and P/CAF for its phosphorylation and acetylation, respectively. USF also interacts with BAF60c which is first phosphorylated by aPKC upon feeding/insulin treatment, bridging other BAF subunits to form LipoBAF complex for chromatin remodeling for activation of the FAS gene. We showed this common mechanism governs transcription of FAS and various other lipogenic genes. Recently, we found a Mediator component, Med17 to interact with USF, which links USF to the preinitiation complex formation for FAS transcription. Furthermore, we started to examine key epigenetic regulation via dynamic CpG and histone demethylation for the transcriptional activation of FAS upon feeding/insulin treatment. We propose following specific aims in this application: Aim 1 is to characterize Med17 interaction with USF and to examine its role in the FAS promoter activation. The significance of feeding/insulin-dependent Med17 phosphorylation will also be studied. Aim 2 is to examine feeding/insulin dependent CpG demethylation at the FAS promoter region and the role of TET in demethylation for lipogenic gene activation. Aim 3 is to examine the function and phosphorylation of the histone demethylase that we found to interact with USF for histone modification and lipogenic gene activation. Finally, Aim 4 is to examine the in vivo roles in lipogenesis by adenovirus mediated shRNA knockdown and overexpression of these enzymes and their phosphorylation mutants in livers of mice. We will study the metabolic consequences, such as hepatic lipogenesis, triglyceride levels, blood lipid levels, and lipid metabolism, which may affect insulin sensitivity. Overall, this research will shed light on how lipogenic genes are activated by feeding/insulin treatment. It may also provide new therapeutic target(s) for metabolic diseases associated with dysregulation of fat metabolism, such as obesity, hepatosteatosis, and diabetes.